A Novel Methylation PCR that Offers Standardized Determination of FMR1 Methylation and CGG Repeat Length without Southern Blot Analysis

Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%–100%) with an accuracy of 99% (95% CI: 93%–100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories.Diverse developmental, mental, and reproductive disorders are associated with both the number of cytosine-guanine-guanine (CGG) repeats and the methylation status of the fragile X mental retardation-1 (FMR1, {"type":"entrez-nucleotide","attrs":{"text":"NM_002024.4","term_id":"169881241","term_text":"NM_002024.4"}}NM_002024.4) gene.^1–3 Excessive CGG repeat expansion is directly linked with hypermethylation of the gene through an epigenetic mechanism that is distinct from X chromosome inactivation and developmentally timed after lyonization.⁴ Because the FMR1 protein (FMRP) is a master regulator of genes involved in synaptic plasticity,⁵ the intellectual and behavioral consequences of quantitative FMR1 silencing are profound. Methylation of full mutation expansions (>200 CGG), however, can be incomplete, and less severe phenotypes may be associated with methylation mosaicism.^6–8 In premutation alleles (55 to 200 CGG) the number of CGG can influence the risks and phenotype of fragile X–associated tremor/ataxia syndrome (FXTAS, OMIM 300623),⁹ fragile X–associated primary ovarian insufficiency (FXPOI, OMIM 300624),^10,11 and autism spectrum disorders.^12,13 Methylation status or X-inactivation in females may further influence the risk and phenotype of these conditions even if the results reported are still inconclusive.^10,11,14 These premutation alleles are relatively common in the general population, occurring in 1 in 130 to 250 women and in 1 in 250 to 810 men, as reported in the United States,^15,16 suggesting a broader need for FMR1 characterization in the general population. Differences in methylation status have also been reported between DNA from whole blood compared to skin fibroblasts, which may be closer in cellular origin to brain and more reflective of phenotype.¹⁷ Thus, it is critical to accurately and reliably assess the CGG repeat length and spectrum of methylation characteristics in individuals with FMR1 premutation and full mutation expansions, and to enable analysis of alternative sample types rather than peripheral blood.Southern blot (SB) analysis is currently the gold standard method for determining size and methylation status in expanded FMR1 alleles. However, this procedure is severely limited by the amount of genomic DNA material that is required, a tedious workflow, and variable sensitivity. Disadvantages of SB include low resolution and the inability to accurately size premutation and normal alleles. Therefore, most clinical laboratories currently rely on a combination of PCR and SB analysis because of the technical limitations and the specific pitfalls of each method that, if used alone, could induce potential misinterpretation of the genotype.^18,19Various alternatives to SB analysis have been reported that use bisulfite or enzymatic pretreatment of DNA before PCR to obtain methylation status.^20–25 These methods have been typically restricted to the analysis of male samples because of the inefficiency of PCR or confounding presence of two X chromosomes in females.^24,25 Alternative methylation markers have been proposed but lack direct association with the number of CGG repeats.²⁶ Concurrent assessment of CGG repeat length and of allele-specific methylation status in both males and females has been demonstrated.²³ This methylation PCR (mPCR) method was based on the analysis of DNA treated with methylation-sensitive endonucleases before FMR1 gene-specific PCR.²⁷ The results were concordant with SB analysis across a range of genotypes. However, a reference control was incorporated that overlapped with samples having alleles of 38 to 42 CGG, and the approach lacked additional controls that would benefit routine testing in a clinical laboratory environment.Herein, we report the inclusion of novel procedural controls and a simplified workflow for mPCR that advance FMR1 analysis without the need for SB analysis. We compare results between methods using a range of challenging clinical samples obtained from two European laboratories. We demonstrate concordance and improved detection of methylation and size mosaicism relative to SB analysis. The ability to analyze novel sample types and samples with aneuploidy that might be encountered during standard fragile X testing is presented. Consequently, this report provides the first interlaboratory validation of a PCR-based FMR1 assay that can accurately assess repeat length and methylation status in both males and females (including all premutation and full mutation alleles), and thus support routine fragile X testing without the requirement for SB analysis.     

Ventilatory response to carbon dioxide and to isometric muscle contraction after administration of placebo, propranolol, mepindolol and salbutamol

Healthy males (23-29 years) volunteered as subjects. We studied the ventilatory response to carbon dioxide and to isometric exercise (50% of maximum voluntary contraction) after administration of propranolol (20 mg), mepindolol (5 mg), salbutamol (20 mg) and placebo (single dose orally). The increase of pulmonary ventilation (VE) activated by central (CO2) and reflex stimulus (hand-grip) did not differ statistically between the four drugs. Analysis of VE in terms of inspiratory drive (Vt/Ti) and timing ratio (Ti/Ttot) showed that during CO2 stimulation mepindolol and salbutamol increased VE predominantly by the increase of Vt/Ti, presumably through the direct stimulation on the respiratory center. During rebreathing, Ti/Ttot increased significantly after administration of placebo and propranolol, so VE increased by a rise of Vt/Ti and by an increase of an effective 'timing component' (Ti/Ttot). Propranolol does not modify the ventilatory response to CO2 and hand-grip when VE is analyzed in terms of Vt/Ti and Ti/Ttot.     

Entropy of dialogues creates coherent structures in e-mail traffic

We study the dynamic network of e-mail traffic and find that it develops self-organized coherent structures similar to those appearing in many nonlinear dynamic systems. Such structures are uncovered by a general information theoretic approach to dynamic networks based on the analysis of synchronization among trios of users. In the e-mail network, coherent structures arise from temporal correlations when users act in a synchronized manner. These temporally linked structures turn out to be functional, goal-oriented aggregates that must react in real time to changing objectives and challenges (e.g., committees at a university). In contrast, static structures turn out to be related to organizational units (e.g., departments).     

Noninvasive risk stratification in arrhythmogenic right ventricular cardiomyopathy

The natural history of arrhythmogenic right ventricular cardiomyopathy is determined by the electrical instability of the dystrophic myocardium, which can precipitate arrhythmic cardiac arrest any time during the course of the disease and by the progressive myocardial loss that results in ventricular dysfunction and heart failure. Sudden death accounts for the majority of the fatal events but its occurrence is mostly unpredictable. There are no prospective and controlled studies assessing clinical markers that can predict the occurrence of life-threatening ventricular arrhythmias. However, the noninvasive risk profile, which emerges from retrospective analysis of clinical and pathologic series, is characterized by history of syncope, physical exercise, spontaneous ventricular tachycardia or ventricular fibrillation, right ventricular dysfunction, left ventricular involvement, right precordial negative T wave, right bundle branch block, QT-QRS dispersion, right precordial ST-segment elevation and late potentials. At present only QRS dispersion, history of syncope and right and/or left ventricular abnormalities at radionuclide angiography proved to be independent noninvasive predictors of sudden death.